In Acanthamoeba castellanii lysosomal enzymes are secreted and accumulate in the growth medium. We have previously shown that the secretion rates of eight enzymes fall into two groups: one about 17% of total cell activity per hour and another at about 3%. The lysosomal enzymes appear to be secreted coincident with membrane recycling from the digestive vacuoles. In order to obtain information about the path of hydrolases to internal compartments as well as their secretion route, we are making antibodies to two lysosomal hydrolases to use as markers in immunocytochemical studies. The methods that we have employed include column chromatography, polyacrylamide gel electrophoresis, and light and electron microscopy. We have isolated and partially purified one enzyme from each secretion group: beta-D-hexosaminidase from the high secretory rate group, and beta-D-glucosidase from the low secretory rate group. Hexosaminidase, purified to near homogeneity by a combination of ion exchange and affinity chromatography, was extracted from SDS gels, deglycosylated and injected into rabbits. Two antibodies with different specificities were obtained, one localizing at least two lysosomal hydrolases and the other specific for beta-hexosaminidase. These antibodies are being used for enzyme localizations at the light and electron microscopic level. Beta-glucosidase, present in lower specific activity in cells, has been partially purified, has an isoelectric point of 5.3 and an apparent molecular mass of 80-Kda from gel permeation columns. The deglycosylated, purified enzyme will be used to prepare antibodies in goats for double label localization of both enzymes in order to study the enzyme secretion route.